The present invention relates to biological diagnostic techniques, and more specifically to the separation and the qualitative and quantitative analysis of microRNAs in human serum or plasma, and to clinical indicators of non-small cell lung cancer. More particularly, the invention relates to a method for detecting and analyzing variations in microRNAs in human serum or plasma. This enables in vitro diagnosis of non-small cell lung cancer; evaluation of disease stage; monitoring of the progression of non-small cell lung cancer; evaluation of disease complications and relapse; prognosis of non-small cell lung cancer; and evaluation of drug efficacy and therapeutic effects.
Non-small cell lung cancer is the most common type of lung cancer, which comprises of 85% of all lung cancer cases. The accurate location and precise detection of non-small cell lung cancer markers is an important precondition for early diagnosis and treatment of non-small cell lung cancer.
Although many disease markers have been discovered and applied in the general survey and diagnosis of clinical diseases and in the monitoring of therapeutic efficacy, obvious deficiencies exist in their clinical application. For instance, the tumor markers that have been widely used in clinical diagnosis, including alpha-fetoprotein (AFP), lactic dehydrogenase (LHD) and carcinoembryonic antigen (CEA), are inadequate for the early stage diagnosis of cancers for various reasons. For example, the sensitivity and specificity of the disease markers mentioned above are relatively low, so that the detection results cannot be used as accurate indicators for disease diagnosis. Furthermore, the disease markers mentioned above cannot satisfy the requirement for early stage diagnosis, which often bears a positive correlation with the therapeutic efficacy. Due to drawbacks including the extremely high specificity of cancer differentiation types, the relatively low sensitivity of tumor bodies, the difficulty of taking repetitive samples, the high requirements for preserving samples, and the expensive costs, the application of the currently known tumor markers cannot be widely promoted under present conditions. Meanwhile, some conventional medical methods such as biopsy for tissue and cell detection have inherent deficiencies, such as taking sample from an incorrect location, insufficiency of tissue and cell sample materials, or the technician's lack of experience, which may all contribute to misdiagnosis. Other techniques like medical imaging that have been widely used in disease inspection and diagnosis are rather limited in the qualitative description of disease process. Therefore, it is necessary to find new types of disease markers that are sensitive and convenient in application, and capable of overcoming the deficiencies of the currently known markers.
Micro ribonucleic acids (microRNAs or miRNAs) are a class of non-coding, single strand, small ribonucleic acid molecules having a length of 19 to 23 nucleotides. They are highly conserved in evolution and closely related to many normal biophysical activities of animals, including ontogenesis, tissue differentiation, apoptosis and energy metabolism, and to the occurrence and progression of many diseases. Recent research has found that the expression level of several microRNAs in chronic lymphocytic leukemia and Burkett lymphoma patients is down-regulated to varying extents (Lawrie C H, Gal S, Dunlop H M et al. Detection of elevated levels of tumor-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br J Haematol 2008; 141:672-675); the analysis of the expression of microRNAs in human lung cancer and breast cancer tissues shows that the expression level of some tissue-specific microRNAs is different from that of normal tissues (Garofalo M, Quintavalle C, Di Leva G et al. MicroRNA signatures of TRAIL resistance in human non-small cell lung cancer. Oncogene 2008). There is also research proving that microRNAs influence the occurrence and progression of angiocardiopathies including cardiac hypertrophy, heart failure and atherosclerosis, and are closely related to metabolic diseases like diabetes type II (Tryndyak V P, Ross S A, Beland F A, Pogribny I P. Down-regulation of the microRNAs miR-34a, miR-127 and miR-200b in rat liver during hepatocarcinogenesis induced by a methyl-deficient diet. Mol Carcinog. 2008 Oct. 21). All of these studies conclude that the expression and specific variations of microRNAs are related to the occurrence and progression of diseases.
MicroRNAs play a highly important role in post-transcription gene regulation, which indicates a relationship between microRNAs and diseases. Firstly, the variations of microRNAs may be the cause of the diseases, because the disease inhibition and promotion factors are probably the targets of microRNAs. MicroRNA expression disorders, for example, down-regulation of genes expressing microRNAs that inhibit the disease promotion factors, or up-regulation of genes expressing microRNAs that inhibit the disease inhibition factors, would lead to down-stream gene expression variation and even lead to some gene-pathway disorders, eventually causing diseases. Secondly, microRNA variations are probably the result of the diseases. Disease (cancers, for instance) may cause loss of chromosome sections, genetic mutation or sudden amplification of chromosome sections; if the microRNAs are from the varied sections, the expression level of these microRNAs will be tremendously altered. Therefore, theoretically, microRNAs can be used as a novel disease marker; the specific alteration is undoubtedly related to disease occurrence and development. Meanwhile, microRNAs may be potential drug target sites; the occurrence and progression of the diseases may be largely relieved by inhibiting up-regulated microRNAs or over-expressing down-regulated microRNAs during the disease progression.
Presently, research on microRNAs as disease markers has been carried out in China. For instance, Chinese patent applications CN100999765A and CN101298630A have disclosed respectively that, in the case of colon cancer, specific variations have occurred to some microRNAs when benign colon polyps deteriorate into malignant tumor. A more sensitive and accurate early-phase colon cancer diagnosis method has been developed according to the specific variations of microRNAs. However, the wide clinical application is restrained by difficulties in obtaining tissue samples.